Epstein-Barr Virus (EBV) is a ubiquitous human gammaherpesivrus that has been classified by the WHO as a class I carcinogen due to its consistent association with lymphoid and epithelial cell malignancies. EBV-associated tumors harbor latent viral genomes that persist as multicopy, chromatin-associated episomes. EBNA1 is the one viral protein known to be essential for episome maintenance and is the only viral protein consistently expressed in all EBV-associated tumors. EBNA1 is a multifunctional, sequence-specific DNA-binding protein that regulates viral DNA replication, episome maintenance, metaphase chromosome tethering, and transcription. EBNA1 is also required for host-cell survival. The molecular mechanisms for each of these functions are only partly understood, and more complete understanding is necessary for development of effective strategies to treat EBV latent infection and carcinogenesis. During the previous funding cycle, we showed that EBNA1 functions at the viral origin of plasmid DNA replication (OriP) through interactions with several host proteins important for chromosome integrity, including telomere repeat binding factors (TRFs), DNA replication pausing factors (Timeless and Tipin), and DNA recombination proteins (MRE11-RAD50-NBS1 or MRN). We have also shown that EBNA1 interacts with the origin recognition complex (ORC) in an RNA-dependent manner and that this correlates with metaphase chromosome attachment. To understand EBNA1 interactions with the host-chromosome, we used chromatin immunoprecipitation combined with next generation DNA sequencing (ChIP-Seq) and functional genomic methods to identify and characterize EBNA1 binding sites in the human genome. We now propose to advance these studies to better understand the role of EBNA1 in viral genome maintenance and host-cell survival during latent infection. We propose to test the overarching hypothesis that EBNA1 coordinates multiple aspects of viral genome persistence with host-cell fitness. We will test the specific hypotheses that EBNA1 (1) promotes recombination-dependent replication at OriP, (2) coordinates viral DNA replication and episome maintenance with telomere length regulation, and (3) modulates cellular gene expression and chromosome structures necessary for host-cell and viral episome survival during latent infection. Since EBNA1 is a prototypical genome maintenance protein, our studies will provide novel insight into the genome maintenance mechanisms shared by Kaposi's Sarcoma-Associated Herpesvirus (KSHV) LANA and human papillomavirus (HPV) E2 family members, as well as by cellular factors that protect fragile genetic elements, including telomeres and other repetitive DNA. More detailed understanding of EBNA1 is further justified by its potential value as a target for therapeutic intervention in diseases and cancers associated with EBV latent infection.